This invention relates to novel materials and processes useful in immunoassay testing of thyroxine, henceforth usually referred to as T.sub.4.
In recent years, it has been recognized that hypothyroidism in neonatal subjects is related to a relatively high incidence of mental retardation. For example, different studies have indicated that the incidence of such hypothyroidism is of the order of about 1 in each 5,000 to 10,000 births. However, early recognition of hypothyroidism and consequent treatment therefor can either prevent or ameliorate any mental retardation which might otherwise result. Consequently, large-scale screening of neonatal subjects is being carried out to determine the T.sub.4 level of their blood. One general type of such a test is described by Larsen and Broskin in Pediatric Research, Volume 9, Pages 604-609 in an article entitled "Thyroxine T.sub.4 Immunoassay Using Filter Paper Blood Sample for Screening of Neonates for Hypothyroidism." The test disclosed therein includes use of standard quantities of thyroxine distributed in small areas ("dots") of a filter paper.
These so-called "standard dots" are then used in assay procedures to determine the T.sub.4 value of blood derived samples brought into contact therewith. This kind of test procedure is convenient to use for large-scale testing. Nevertheless, there have been substantial problems involved in obtaining inexpensive, dependable, easily absorbed, easily wetted, and easily processed standards.
The test employs the application of radioimmunoassay techniques to assess the concentration of neonatal thyroxine (T.sub.4). The essential elements of this procedure are:
1. The development of an antibody, specific for T.sub.4 and without significant cross reactivity with other substances. PA1 2. Competition for a limited number of antibody binding sites between a radio-active isotope of T.sub.4 and normal T.sub.4. PA1 3. separation and determination of the two components once equilibrium has occurred. PA1 Ag represents Unlabeled antigen (standard or unknown) PA1 Ab represents the specific antibody PA1 Ag-Ab represents unlabeled antibody-antigen complex PA1 Ag*-Ab represents labeled antibody-antigen complex.
In practice, known amounts of T.sub.4 (standards) and unknown quantities of T.sub.4 (samples), antiserum specific for T.sub.4, and .sup.125 1-T.sub.4 are incubated together. Once equilibrium is established the free hormone is separated from the hormone bound to the antiserum by the adsorption of the free to dextran-coated charcoal. The amount of bound .sup.125 1-T.sub.4 present in the supernatant is determined with a gamma counter. Because stable T.sub.4 is always present in greater concentration, the binding of T.sub.4 1.sup.125 to the antibody will be inhibited and the degree of inhibition will be proportional to the amount of stable T.sub.4.
A standard curve can be established by measuring the fraction of antibody-bound .sup.125 1-T.sub.4 after the addition of known concentrations of stable T.sub.4.
The reaction can be represented by the equation: ##STR1## Where Ag* represents labeled antigen (hormone)
In general, it is intended that the estimation of T.sub.4 concentration be carried out in the capillary blood of neonatal subjects only. It is most advantageous as a preliminary diagnostic qualifying test. Results indicative of hypothyroidism should be combined with a TSH determination.
Although not directly relevant to the precise invention disclosed herein, a substantial amount of prior art relating to various immunoassay tests for T.sub.4 is described in various U.S. patents classified in Class 424-1 of the U.S. Patent Office Manual of Classification.